DNA encoding 2-acyltransferases

ABSTRACT

Plants, particularly transgenic plants, may be produced having a 2-acyltransferase enzyme or other insoluble acyltransferase enzyme with an altered substrate specificity compared to the native enzyme. For example, oil seed rape (Brassica napus) may contain a 2-acyltransferase transgene derived from Limnanthes douglassi in order to increase the erucic acid content of the oil. The cDNA sequence of maize (Zea mays) 2-acyltansferase is disclosed and is useful for cloning acyltransferase genes and/or cDNAs from other organisms, including L. douglassi.

FIELD OF THE INVENTION

This invention relates to modified plants. In particular, the inventionrelates to plants modified such that at least part of the plant (forexample seeds of the plant) is capable of yielding a commercially usefuloil.

BACKGROUND OF THE INVENTION

Plants have long been a commercially valuable source of oil. Nutritionaluses of plant-derived oils have hitherto been dominant, but attention isnow turning additionally to plants as a source of industrially usefuloils, for example as replacements for or improvements on mineral oils.Oil seeds, such as from rape, have a variety of lipids in them (Hildish& Williams, "Chemical Composition of Natural Lipids", Chapman Hall,London, 1964). There is now considerable interest in altering lipidcomposition by the use of recombinant DNA technology (Knauf, TIBtech,February 1987, 40-47), but by no means ail of the goals have beenrealised to date for a variety of reasons, in spite of theever-increasing sophistication of the technology.

Success in tailoring the lipid content of plant-derived oils requires afirm understanding of the biochemistry and genes involved. Broadly, twoapproaches are available. First, plants may be modified to permit thesynthesis of fatty acids which are new (for the plant); so, for example,laurate and/or stearate may be synthesised in rape. Secondly, thepattern and/or extent of incorporation of fatty acids into the glycerolbackbone of the lipid may be altered. It is with this latter approachthat the present invention is concerned, although the former approachmay additionally be used.

Lipids are formed in plants by the addition of fatty acid moieties ontothe glycerol backbone by a series of acyl transferase enzymes. There arethree positions on the glycerol molecule at which fatty acid (acyl)moieties may be substituted, and the substitution reached at eachposition is catalysed by a position-specific enzyme: the enzymes areknown as 1-, 2- and 3-acyltransferases, respectively.

One, but not the only, current aim of "lipid engineering" in plants isto provide oils including lipids with a high content of erucic (22:1)acid. Erucic acid-containing lipids are commercially desirable for anumber of purposes, particularly as replacements to or supplements formineral oils in certain circumstances, as alluded to above. In the caseof oil seed rape (Brassica napus), one of the most significant oilproducing crops in cultivation today, the specificity of the2-acyltransferase enzyme positively discriminates against theincorporation of erucic acid at position 2. So, even in those cultivarsof rape which are able to incorporate erucic acid at positions 1 and 3,where there is no (or at least reduced) discrimination against erucicacid, only a maximum 66% of the fatty acids incorporated into triacylglycerols can be erucic acid. Such varieties of rape are known as HEAR(high erucic acid rape) varieties.

It would therefore be desirable to increase the erucic acid content ofconventional oil seed rape, as well as HEAR varieties; the same can besaid of oils of other vegetable oil crops such as maize, sunflower andsoya, to name but a few examples. While in principle it may be thoughtpossible to introduce into a desired plant DNA encoding a2-acyltransferase of different fatty acid specificity, for example froma different plant, in practice there are a number of problems.

First, 2-acyltransferase and 3-acyltransferase are membrane bound, andtherefore insoluble, enzymes. They have not been purified. This makesworking with them difficult and rules out the use of many conventionalDNA cloning procedures. This difficulty does not, paradoxically, lie inthe way of cloning the gene (or at least cDNA) encoding the1-acyltransferase enzyme, which is soluble: in fact, recombinant DNAwork has already been undertaken on this enzyme for a completelydifferent purpose, namely the enhancement of chilling resistance intobacco plant leaves, by Murata et al (Nature 356 710-713 (1992)).

Secondly, very little is known about the 2- and 3-acyltransferases.There is no idea of their size or how they are targeted to membranes. Nonucleotide or amino acid sequence data are available and no antibodieshave been raised against them.

Although there has been discussion, therefore, of the desirability ofmodifying 2-acyltransferase specificity, for example by importing a genecoding for the corresponding enzyme, but of different specificity, fromanother species, there is a pressing need in the art for the key whichenables this work to be done.

SUMMARY AND DETAILED DESCRIPTION OF THE INVENTION

The present invention provides such a key, in the form of a DNA sequence(in the specific case, a cDNA sequence) encoding a 2-acyltransferase.The DNA sequence in FIGS. 1A-1C (SEQ ID NO: 1) from nucleotides 130 to1254 encodes the 2-acyltransferase from maize (Zea mays), including thestop codon.

According to a first aspect of the invention, therefore, there isprovided a recombinant or isolated DNA sequence, preferably encoding anenzyme having membrane-bound acyltransferase activity, and selectedfrom:

(i) a DNA sequence comprising the DNA sequence of FIGS. 1A-1C (SEQ IDNO: 1) encoding at least from MET₁ to Stop₃₇₅ (SEQ ID NO: 2) or itscomplementary strand,

(ii) nucleic acid sequences hybridising to the DNA sequence of FIGS.1A-1C (SEQ ID NO: 1), or its complementary strand, under stringentconditions, and

(iii) nucleic acid sequences which would hybridise to the DNA sequenceof FIGS. 1A-1C (SEQ ID NO: 1)(SEQ ID NO: 10), or its complementarystrand, but for the degeneracy of the genetic code.

Fragments of the above DNA sequences, for example of at least 15, 20,30, 40 or 60 nucleotides in length, are also within the scope of theinvention.

Suitable stringent conditions include salt solutions of approximately0.9 molar at temperatures of from 35° C. to 65° C. More particularly,stringent hybridisation conditions include 6× SSC, 5× Denhardt'ssolution, 0.5% SDS, 0.5% tetrasodium pyrophosphate and 50 μg/mldenatured herring sperm DNA; washing may be for 2×30 minutes at 65° C.in 1× SSC, 0.1% SDS and 1×30 minutes in 0.2× SSC, 0.1% SDS at 65° C.

Nucleic acid sequences within the scope of the first aspect of theinvention will generally encode a protein having 2-acyltransferaseactivity, as that is the activity of the enzyme encoded by the nucleicacid sequence of FIGS. 1A-1C (SEQ ID NO: 1). Nucleic acid sequences notencoding a protein having enzymic activity (or the relevant enzymicactivity) but otherwise conforming to the first aspect of the inventionas set out above may be useful for other purposes (and are thereforealso encompassed by the invention); for example they may be useful asprobes, which is a utility shared by the nucleic acid sequences of thefirst aspect of the invention, including the FIG. 1 sequence itself.

The probe utility arises as follows. As there is likely to be a highdegree of homology between acyltransferases of different species (andparticularly between 2-acyltransferases of different species) thesequence of FIGS. 1A-1C (or part of it, or other sequences within theinvention) may be used to probe cDNA or genomic libraries of otherspecies in order to clone DNA sequences encoding acyltransferases havingdesired specificities. For example, if it is desired to produce oilhaving a high content of erucic acid esterified to glycerol, a DNAlibrary of any species which naturally makes erucic acid may be probed.Suitable plants include meadow foam (Limnanthes spp., especially L. albaand, particularly, L. douglassi) and Crambe. Limnanthes douglassi is thepreferred species, as specificity studies show that there is positivediscrimination towards incorporation of erucic acid into position 2 ofthe triacylglyceride. Libraries of organisms other than the higherplants may be probed; for example, certain bacteria may have anacyltransferase of the desired specificity.

DNA in accordance with the invention will in general have a higherdegree of homology with at least part of the sequence FIGS. 1A-1C (SEQID NO: 1) than with known sequences.

Recombinant DNA in accordance with the invention may be in the form of avector, which may have sufficient regulatory sequences (such as apromoter) to direct expression. Vectors which are not expression vectorsare useful for cloning purposes (as expression vectors themselves maybe) . Host cells (such as bacteria and plant cells) containing vectorsin accordance with the invention themselves form part of the invention.

DNA sequences in accordance with the invention can be used in anotherway in cloning a gene of interest from another species: if the DNA iscoupled to a suitable promoter, for example on an expression vector in asuitable host organism, protein may be produced. Such protein may beused to generate polyclonal or monoclonal antibodies, or other bindingmolecules, which may then be used to screen for expression of homologousproteins in other species, for example as part of a DNA libraryscreening programme.

Suitable CDNA libraries of.target species will generally be preparedwhen the gene of interest is likely to be expressed; so cDNA embryolibraries (prepared at the early lipid synthesis stage), for example ofLimnanthes spp. will be preferred.

The invention therefore enables the cloning of a wide variety of genes(or, more generally, DNA sequences) encoding acyltransferases, and2-acyltransferases in particular, using DNA sequences as describedabove.

Such acyltransferases, such as from Limnanthes spp. may also be cloneddirectly, for example using complementation studies, from a DNA libraryof the species in question. For example, if E. coli is used as thecomplementation host, a mutant is chosen which is defective in therelevant enzyme (for example 2-acyltransferase); the DNA library fromthe target species (such as L. douglassi) is cloned into the mutantcomplementation host; host cells incorporating the targetacyltransferase gene in their genome can readily be selected usingappropriate selective media. E. coli mutant JC201 is a suitable host foruse in complementation studies relating to 2-acyltransferase.

Cloning the acyltransferase gene of choice into a microbial host, suchas a bacterium like E. coli, in such a way that the gene can beexpressed has a particularly advantage in that the substrate specificityof the acyltransferase gene can be assessed in the microbial host beforetransformed plants are prepared, thereby saving considerably on researchtime. Such an assessment may be made by competitive substrate assays, inwhich differently detectably labelled candidate substrates for theenzyme compete with each other for incorporation into the glyceride. Forexample, ¹⁴ C-erucyl CoA and ³ H-oleoyl CoA can be used as competitivesubstrates for 2-acyltransferase, and the relative amounts of ¹⁴ C ortritium uptake into glyceride can be measured. (As 2-acyltransferaseshave acceptor, glycerol-based, substrates and donor, fatty acid-based,substrates, the experiment can be carried out with different acceptors,such as 1-erucyl-glycerol-3-phosphate and1-oleoyl-glycerol-3-phosphate.) A gene coding for an enzyme whichpreferentially donates erucic acid to the acceptor (particularly1-erucyl-glycerol-3-phosphate) may by this means be identified as a DNAsequence of choice for further use in the invention as described below.

In a second aspect of the invention, there is provided a plant havingone or more insoluble acyltransferase enzymes having a substratespecificity which differs from the native enzyme of the plant.

While site-directed mutagenesis and/or other protein engineeringtechniques may be used to alter the specificity of an enzyme native tothe plant, it is preferred that the plant be transgenic and incorporatean expressible acyltransferase gene encoding an enzyme of the desiredspecificity from another species. 2-acyltransferases are the enzymes ofchoice. For example, as described above, a 2-acyltransferase enzymewhich has an enhanced specificity for, or at least no discriminationagainst, erucic acid, may be made by this means to express in a plantwhich would not normally incorporate erucic acid into triacylglycerides.An important embodiment of the invention relates to geneticallyengineered plants which have higher levels of erucic acid incorporatedinto triacylglycerols than in corresponding non-engineered plants.Preferable though this embodiment may be, though, the invention is notlimited to the enhancement of erucic acid incorporation into glycerides:other acids may be desired in other circumstances.

For the acyltransferase transgene to be expressible, a promoter has tobe operatively coupled to it. Because at the present state of the art itis difficult precisely to regulate the site of incorporation of atransgene into the host genome, it is preferred that the transgene becoupled to its promoter prior to transformation of the plant. Promotersuseful in the invention may be temporal- and/or seed-specific, but thereis no need for them to be so: constitutive promoters, such as the CaMV35S promoter, may be in fact be preferred because they are usuallystrong promoters. Other tissues are unlikely to be adversely affected ifthe transgene encoding the acyltransferase enzyme is expressed in them,as the availability of the fatty acid CoA substrates is effectivelylimited to the seed.

The promoter-transgene construct, once prepared, is introduced intoplant cells by any suitable means. The invention extends to such plantcells. Preferably, DNA is transformed into plant cells using a disarmedTi-plasmid vector and carried by Agrobacterium by procedures known inthe art, for example as described in EP-A-0116718 and EP-A-0270822.Alternatively, the foreign DNA could be introduced directly into plantcells using an electrical discharge apparatus. This method is preferredwhere Agrobacterium is ineffective, for example where the recipientplant is monocotyledonous. Any other method that provides for the stableincorporation of the DNA within the nuclear DNA of any plant cell of anyspecies would also be suitable. This includes species of plant which arenot currently capable of genetic transformation.

Preferably DNA in accordance with the invention also contains a secondchimeric gene (a "marker" gene) that enables a transformed plant ortissue culture containing the foreign DNA to be easily distinguishedfrom other plants or tissue culture that do not contain the foreign DNA.Examples of such a marker gene include antibiotic resistance(Herrera-Estrella et al, EMBO J. 2(6) 987-95 (1983) and Herrera-Estrellaet al, Nature 303 209-13 (1983)), herbicide resistance (EP-A-0242246)and glucuronidase (GUS) expression (EP-A-0344029). Expression of themarker gene is preferably controlled by a second promoter which allowsexpression in cells other than the tapetum, thus allowing selection ofcells or tissue containing the marker at any stage of regeneration ofthe plant. The preferred second promoter is derived from the gene whichencodes the 35S subunit of Cauliflower Mosaic Virus (CaMV) coat protein.However any other suitable second promoter could be used.

A whole plant can be regenerated from a single transformed plant cell,and the invention therefore provides transgenic plants (or parts ofthem, such as propagating material) including DNA in accordance with theinvention as described above. The regeneration can proceed by knownmethods.

In one embodiment of the invention, the transgenic plant's nativeacyltransferase gene which corresponds to the transgene may be renderedat least partially inoperative or removed. So, if the transgene encodesa 2-acyltransferase, the plant's native 2-acyltransferase may berendered inoperative by, for example, antisense or ribozyme techniques,as is known in the art.

By means of the invention, plants generating oil with a tailored lipidcontent may be produced. For example, the lipid composition oftriacylglycerides in a plant may be substantially altered to producetriacylglycerides with a desired fatty acid (for example erucic acid)content higher than has hitherto been possible. For example, oil seedrape (B. napus) may be transformed to produce oil whose triacylglyceridehas an erucic acid content of over 70%.

It can readily be seen that plants with increased lipid levels may beproduced by means of the invention. However, the invention is alsouseful for producing plants with decreased lipid levels, which may bedesired if elevated protein and/or starch levels are required. Decreasedlipid levels may be achieved by interfering with the proper functioningof a gene encoding a 2-acyltransferase, for example by antisense orribozyme technology. (Such reduced-lipid plants may if desired befurther engineered for higher protein and/or starch content, if wished.)

Promoters which naturally drive 2-acyltransferases may also be obtainedby hybridisation and/or restriction and/or sequencing studies using thesequence of FIGS. 1A-1C.

The invention enables the production of protein encoded by DNA of thefirst aspect of the invention, should that be desired. The protein maybe expressed by host cells harbouring DNA in the form of an expressionvector. The protein, which may be an enzyme having 2-acyltransferaseactivity, may have an amino acid sequence which is identical to orhomologous with the sequence of FIGS. 1A-1C (SEQ ID NO: 2). The degreeof homology will generally be greater than that of known proteins, andmay be at least 40, 50, 60, 70, 80, 90, 95 or 99%.

Preferred features of each aspect of the invention are as for each otheraspect mutatis mutandis.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated by the following examples. The examplesrefer to the accompanying drawings, in which:

FIGS. 1A-1C shows the cDNA sequence derived in Example 1 (SEQ ID NO: 1)and its derived protein sequence (SEQ ID NO: 2).

FIG. 2 shows a sequence alignment of part of the gene products of plsB(SEQ ID NO: 3) and plsC (SEQ ID NO: 4) with part of the sequence shownin FIGS. 1A-1C (SEQ ID NO: 5), showing a conserved motif. plsb is the E.coli sn-glycerol-3-phosphate acyltransferase gene and plsC the1-acyl-sn-glycerol-3-phosphate acyltransferease gene of E. coli. Doublepoints indicate exact matches between two sequences and a single pointconservative amino acid substitutions. Stars indicate identical aminoacids in all three sequences and residues conserved in two out of thethree sequences are marked by a + symbol.

FIGS. 3A, 3B and 3C: Membrane phospholipids from E. coli strains wereextracted into chloroform and separated by 2-dimensional thin layerchromatography. The first dimension (ascending) was developed usingchloroform:methanol:water (65:25:4) and the second dimension (left toright) developed with chloroform:methanol:acetic acid (65:25:10).Phospholipids were visualised by autoradiography for 16 hours at -70° C.using Fuji RX film. The E. coli strains used were (FIG. 3A): JC201 whichcarries a thermosensitive mutation in the 1-acyl-sn-glycerol-3-phosphateacyltransferase gene; (FIG. 3B): JC201 containing the plasmid pPLSC,which encodes the E. coli 1-acyl-sn-glycerol-3-phosphate acyltransferasegene; (FIG. 3C): JC201 containing the plasmid whose cDNA insert sequenceis shown in FIGS. 1A-1C. LPA, lysophosphatidic acid; PE,phosphatidylethanolamine; CL, cardiolipin; PG, phosphatidylglycerol; PA,phosphatidic acid; O, origin. 20% of the ³² P is incorporated in LPA inJC201 and all of the corresponding label is incorporated in PE in bothof the other two strains.

FIG. 4: Acyltransferase assays were performed using ³² P-labelledlysophosphatidic acid which had been extracted from the E. coli strainJC201 and oleoyl CoA as an acyl donor. Phospholipids present in thereaction mixtures were extracted into chloroform and separated usingsilica gel thin layer chromatography. Chloroform:methanol:aceticacid:water (25:15:4:2) was used to develop the plates. The phospholipidswere visualised by autoradiography for 16 hours at -70° C. using Fuji RXfilm. The E. coli strains used were: JC201 which carries athermosensitive mutation in the 1-acyl-sn-glycerol-3-phosphateacyltransferase gene; JC201 containing the plasmid PPLSC which encodesthe E. coli 1-acyl-sn-glycerol-3-phosphate acyltransferase gene; JC201containing the plasmid whose maize cDNA insert sequence is shown inFIGS. 1A-1C. LPA, lysophosphatidic acid; PA, phosphatidic acid.

FIGS. 5A and 5B shows a comparison of the protein sequence shown inFIGS. 1A-1C (SEQ ID NO: 6) with that derived (SEQ ID NO: 7) from a B.napus seed cDNA insert which was isolated by DNA hybridisation to themaize cDNA sequence. The sequences were aligned with the FastA alignprogram (1988). Double points signify identical amino acids and singlepoints conservative amino acid substitutions.

Maize=374 aa vs. rape=311 aa 51.5% identity; Optimised score: 705

EXAMPLE 1 Derivation of the DNA sequence of FIGS. 1A-1C

Complementation studies using a maize cDNA expression librarytransferred into the E. coli mutant JC201 allowed the isolation of aplasmid encoding a 2-acyltransferase enzyme from maize. The cDNA insertof this plasmid is 1.6 kb in size, and includes a poly A tail of 70 bp.The insert was sequenced to give the data shown and translation of thesequence revealed the present of only one large open reading frame. Thisis shown on FIGS. 1A-1C with proposed start methionine and stop codon inbold print. The 2-acyltransferase is 374 amino acids in size andsequencing upstream of open reading from showed that the protein isexpressed as part of a fusion protein in E. coli. This consists of 10amino acids of the β-galactosidase protein, 43 amino acids (shown insequence) corresponding to the 5' untranslated region of the MRNA andthe 374 amino acid protein. Protein sequence comparisons of the largeopen reading frame with the 2-acyltransferase of E. coli show littleoverall identity but there is a stretch of 80 residues which has a highlevel of conservative substitution and contains some amino acids thatare conserved in the 2-acyltransferase, 1-acyltransferase and N-acetylglucosamine acyltransferase of E. coli.

EXAMPLE 2 Incorporation of 32p into total phospholipids

E. coli strains were grown in minimal medium containing ³² Porthophosphate. Total glycerolipids were extracted into organic solventsand separated by 2D thin layer chromatography (FIG. 3) (Lysophosphatidicacid (LPA) is the substrate for 2-acyltransferase (2-AT)).

As can be seen in FIG. 3A, the accumulation of ³² P-labelled LPA in themutant JC 201 illustrates the absence of a fully functional 2-AT.Addition of a plasmid carrying either the native E. coli gene (FIG. 3B),or the maize clone given in FIGS. 1A-1C (FIG. 3C) restores 2-AT activityto the cells, allowing LPA to be removed and further metabolised.(Lysophosphatidic acid.)

These data indicate that the DNA sequence given in FIGS. 1A-1C codes for2-AT.

EXAMPLE 3 Over expression of the cDNA

The cDNA region specifying the protein sequence given in FIGS. 1A-1C wascloned into the E. coli overexpression vector pET11d (Studier et al,Meth. Enzymol. 185 60-89 (1990)). Increased 2-acyltransferase activityfollowing induction of expression from the plasmid insert confirmed thatthe sequence in FIGS. 1A-1C is that of 2-AT.

EXAMPLE 4 Localisation of 2-AT activity in E. coli cells containing themaize clone

2-acyltransferase assays were carried out using membranes isolated fromthe mutant strain JC.201 which lacks 2-AT and from JC.201 containing themaize plasmid (FIG. 4).

2-AT activity was not detected in membrane fractions from JC.201. Theaddition of a plasmid carrying the native E. coli gene or the sequencegiven in FIGS. 1A-1C (SEQ ID NO: 1), to JC.201 resulted in restorationof 2-AT activity to the membranes.

EXAMPLE 5 Using the maize cDNA as a heterologous probe to obtain cDNAfrom oilseed rape

A seed cDNA library from Brassica napus was screened with the sequencegiven in FIGS. 1A-1C (SEQ ID NO: 1), using standard techniques (Sambrooket al "Molecular Cloning--A Laboratory Manual", 2nd Edition, Cold SpringHarbor Laboratory Press, 1989).

Conditions

For the hybridisation of the maize cDNA insert to the rape library:hybridisation was in 6× SSC, 5× Denhardts solution, 0.5% SDS, 0.5%tetrasodium pyrophosphate and 50 ugml⁻¹ denatured herring sperm DNA. Thefilters were washed 2×30 minutes at 65° C. in 1× SSC, 0.1% SDS and 1×30minutes in 0.2× SSC, 0.1% SDS at 65° C.

A hybridising clone was sequenced and a protein sequence derived for thelarge ORF. Alignment of this protein sequence with that derived from themaize cDNA clone given in FIGS. 1A-1C (SEQ ID NO: 1), is shown in FIGS.5A and 5B.

The strong identity between these sequences illustrates the potential ofusing the sequence given in FIGS. 1A-1C (SEQ ID NO: 1) to obtain other2-ATS.

EXAMPLE 6 Transgenic plants

The sequence given in FIGS. 1A-1C (SEQ ID NO: 1) can be cloned,alongside a suitable promoter, into a suitable vector for expression inplants. The vector can be used to transform plants and the resultingplants expressing the 2-AT can be analysed for lipid content. Lipidmetabolism is expected to be upregulated and elevated lipid levels weredetectable in seeds.

EXAMPLE 7 Antisense

The sequence given in FIGS. 1A-1C (SEQ ID NO: 1) may be cloned,alongside a suitable promoter, in the antisense orientation into asuitable vector for expression in plants. The vector can be used totransform plants and the resulting plants expressing the 2-AT can beanalysed for protein and starch content. Elevated levels of starch andprotein are expected to be detectable in seeds.

EXAMPLE 8 Down-regulation of Native 2-AT

The DNA sequence of a 2-AT derived from L. douglassii (obtained asdescribed in Example 5) can be introduced into oilseed rape (OSR) underthe expression of a suitable promoter, using vectors and planttransformation methods well known in the art. A second sequence,comprising antisense or ribozymes against the rape cDNA (Example 5) canbe introduced for simultaneous expression. The resultant transformedplant is expected to have 2-AT activity corresponding to that of L.douglassii, with concurrent down regulation of the native rape 2-ATgene.

The modified OSR plant thus obtained had higher levels of erucic acid inposition 2 of its triacylglycerols than wild type plants. In additionhigher levels of trierucin are found in the seed oil.

EXAMPLE 9 Genomic library screening

The sequence given in FIGS. 1A-1C (SEQ ID NO: 1) is used to screen agenomic library of Arabidopsis and a hybrid using clone obtained. Usingstandard techniques, a promoter may be derived from this clone. Thepromoter may be used to drive expression in plant cell membranes.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 7                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1514 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 130..1254                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      CCCCGTCCTCCTCGTCGCCGGCGGAGCCGCCTACTATCGCCTGGAGAAGGAGCGCCGCGG60                GGAGCTTTTCCCACTGCCGACTGCCGTCTGACCCTCCGAGATCGGAAGCGGCGCCGGCGC120               CGGCCGGCGATGGCGATCCCGCTCGTGCTCGTCGTGCTCCCGCTCGGC168                           MetAlaIleProLeuValLeuValValLeuProLeuGly                                       1510                                                                          CTGCTCTTCCTCCTGTCCGGCCTCATCGTCAACGCCATCCAGGCCGTC216                           LeuLeuPheLeuLeuSerGlyLeuIleValAsnAlaIleGlnAlaVal                              152025                                                                        CTATTTGTGACGATAAGGCCCTTTTCGAAGAGCTTCTACCGTCGGATC264                           LeuPheValThrIleArgProPheSerLysSerPheTyrArgArgIle                              30354045                                                                      AACAGATTCTTGGCCGAGCTGCTGTGGCTTCAGCTTGTCTGGGTGGTG312                           AsnArgPheLeuAlaGluLeuLeuTrpLeuGlnLeuValTrpValVal                              505560                                                                        GACTGGTGGGCAGGTGTTAAGGTACAACTGCATGCAGATGAGGAAACT360                           AspTrpTrpAlaGlyValLysValGlnLeuHisAlaAspGluGluThr                              657075                                                                        TACAGATCAATGGGTAAAGAGCATGCACTCATCATATCAAATCATCGG408                           TyrArgSerMetGlyLysGluHisAlaLeuIleIleSerAsnHisArg                              808590                                                                        AGTGATATTGATTGGCTCATTGGATGGATATTGGCCCAGCGTTCAGGG456                           SerAspIleAspTrpLeuIleGlyTrpIleLeuAlaGlnArgSerGly                              95100105                                                                      TGCCTTGGAAGTACACTTGCTGTCATGAAGAAGTCATCCAAGTTCCTT504                           CysLeuGlySerThrLeuAlaValMetLysLysSerSerLysPheLeu                              110115120125                                                                  CCAGTTATTGGCTGGTCAATGTGGTTTGCAGAGTACCTCTTTTTGGAA552                           ProValIleGlyTrpSerMetTrpPheAlaGluTyrLeuPheLeuGlu                              130135140                                                                     AGGAGCTGGGCCAAGGATGAAAAGACACTAAAGTGGGGTCTCCAAAGG600                           ArgSerTrpAlaLysAspGluLysThrLeuLysTrpGlyLeuGlnArg                              145150155                                                                     TTGAAAGACTTCCCTAGACCATTTTGGCTAGCTCTTTTCGTCGAGGGT648                           LeuLysAspPheProArgProPheTrpLeuAlaLeuPheValGluGly                              160165170                                                                     ACTCGCTTTACTCCAGCAAAGCTTCTCGCAGCTCAGGAATATGCGGCC696                           ThrArgPheThrProAlaLysLeuLeuAlaAlaGlnGluTyrAlaAla                              175180185                                                                     TCCCAGGGCTTACCGGCTCCTAGAAATGTACTTATTCCACGTACCAAG744                           SerGlnGlyLeuProAlaProArgAsnValLeuIleProArgThrLys                              190195200205                                                                  GGATTTGTATCTGCTGTAAGTATTATGCGAGATTTTGTTCCAGCCATT792                           GlyPheValSerAlaValSerIleMetArgAspPheValProAlaIle                              210215220                                                                     TATGATACAACTGTAATAGTCCCTAAAGATTCCCCTCAACCAACAATG840                           TyrAspThrThrValIleValProLysAspSerProGlnProThrMet                              225230235                                                                     CTGCGGATTTTGAAAGGGCAATCATCAGTGATACATGTCCGCATGAAA888                           LeuArgIleLeuLysGlyGlnSerSerValIleHisValArgMetLys                              240245250                                                                     CGTCATGCAATGAGTGAGATGCCAAAATCAGATGAGGATGTTTCAAAA936                           ArgHisAlaMetSerGluMetProLysSerAspGluAspValSerLys                              255260265                                                                     TGGTGTAAAGACATTTTTGTGGCAAAGGATGCCTTACTGGACAAGCAT984                           TrpCysLysAspIlePheValAlaLysAspAlaLeuLeuAspLysHis                              270275280285                                                                  TTGGCAACAGGCACTTTCGATGAGGAGATTAGACCTATTGGCCGTCCA1032                          LeuAlaThrGlyThrPheAspGluGluIleArgProIleGlyArgPro                              290295300                                                                     GTGAAATCATTGCTGGTGACCCTGTTCTGGTCGTGCCTCCTGCTGTTT1080                          ValLysSerLeuLeuValThrLeuPheTrpSerCysLeuLeuLeuPhe                              305310315                                                                     GGCGCCATCGAGTTCTTCAAGTGGACACAGCTTCTGTCGACGTGGAGG1128                          GlyAlaIleGluPhePheLysTrpThrGlnLeuLeuSerThrTrpArg                              320325330                                                                     GGTGTGGCGTTCACTGCCGCAGGGATGGCGCTTGTGACGGGTGTCATG1176                          GlyValAlaPheThrAlaAlaGlyMetAlaLeuValThrGlyValMet                              335340345                                                                     CATGTCTTCATCATGTTCTCCCAGGCTGAGCGGTCGAGCTCAGCCAGG1224                          HisValPheIleMetPheSerGlnAlaGluArgSerSerSerAlaArg                              350355360365                                                                  GCGGCACGGAACCGGGTCAAGAAGGAATGAAAAATGGAGGGTGGAGA1271                           AlaAlaArgAsnArgValLysLysGlu                                                   370375                                                                        TGAGGTTCTCGTGGGGTTTGTTATGGGCAACCTTCAAAAGGACTCTCCATTCATATTAGT1331              ATTAATTCATATATATGCAGCGCCAAATTCCAGACATTGATATGCTCTCAAATAGGATGT1391              TCTGCTCCCCTCTTGTATTTGTATGCAGGAAAGGGTTTGTAGGGAGTTTACCCCCCCCCC1451              CCCCCCCCCCGCCTTTCTTTGGGGAAGAAAGACATATTCTGGAAGCCTTCCAGTAGTTCA1511              AAA1514                                                                       (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 374 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      MetAlaIleProLeuValLeuValValLeuProLeuGlyLeuLeuPhe                              151015                                                                        LeuLeuSerGlyLeuIleValAsnAlaIleGlnAlaValLeuPheVal                              202530                                                                        ThrIleArgProPheSerLysSerPheTyrArgArgIleAsnArgPhe                              354045                                                                        LeuAlaGluLeuLeuTrpLeuGlnLeuValTrpValValAspTrpTrp                              505560                                                                        AlaGlyValLysValGlnLeuHisAlaAspGluGluThrTyrArgSer                              65707580                                                                      MetGlyLysGluHisAlaLeuIleIleSerAsnHisArgSerAspIle                              859095                                                                        AspTrpLeuIleGlyTrpIleLeuAlaGlnArgSerGlyCysLeuGly                              100105110                                                                     SerThrLeuAlaValMetLysLysSerSerLysPheLeuProValIle                              115120125                                                                     GlyTrpSerMetTrpPheAlaGluTyrLeuPheLeuGluArgSerTrp                              130135140                                                                     AlaLysAspGluLysThrLeuLysTrpGlyLeuGlnArgLeuLysAsp                              145150155160                                                                  PheProArgProPheTrpLeuAlaLeuPheValGluGlyThrArgPhe                              165170175                                                                     ThrProAlaLysLeuLeuAlaAlaGlnGluTyrAlaAlaSerGlnGly                              180185190                                                                     LeuProAlaProArgAsnValLeuIleProArgThrLysGlyPheVal                              195200205                                                                     SerAlaValSerIleMetArgAspPheValProAlaIleTyrAspThr                              210215220                                                                     ThrValIleValProLysAspSerProGlnProThrMetLeuArgIle                              225230235240                                                                  LeuLysGlyGlnSerSerValIleHisValArgMetLysArgHisAla                              245250255                                                                     MetSerGluMetProLysSerAspGluAspValSerLysTrpCysLys                              260265270                                                                     AspIlePheValAlaLysAspAlaLeuLeuAspLysHisLeuAlaThr                              275280285                                                                     GlyThrPheAspGluGluIleArgProIleGlyArgProValLysSer                              290295300                                                                     LeuLeuValThrLeuPheTrpSerCysLeuLeuLeuPheGlyAlaIle                              305310315320                                                                  GluPhePheLysTrpThrGlnLeuLeuSerThrTrpArgGlyValAla                              325330335                                                                     PheThrAlaAlaGlyMetAlaLeuValThrGlyValMetHisValPhe                              340345350                                                                     IleMetPheSerGlnAlaGluArgSerSerSerAlaArgAlaAlaArg                              355360365                                                                     AsnArgValLysLysGlu                                                            370                                                                           (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      TyrPheValGluGlyGlyArgSerArgThrGlyArgLeuLeuAsp                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      MetPheProGluGlyThrArgSerArgGlyArgGlyLeuLeuPro                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      LeuPheValGluGlyThrArgPheThrProAlaLysLeuLeuAla                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 374 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      MetAlaIleProLeuValLeuValValLeuProLeuGlyLeuLeuPhe                              151015                                                                        LeuLeuSerGlyLeuIleValAsnAlaIleGlnAlaValLeuPheVal                              202530                                                                        ThrIleArgProPheSerLysSerPheTyrArgArgIleAsnArgPhe                              354045                                                                        LeuAlaGluLeuLeuTrpLeuGlnLeuValTrpValValAspTrpTrp                              505560                                                                        AlaGlyValLysValGlnLeuHisAlaAspGluGluThrTyrArgSer                              65707580                                                                      MetGlyLysGluHisAlaLeuIleIleSerAsnHisArgSerAspIle                              859095                                                                        AspTrpLeuIleGlyTrpIleLeuAlaGlnArgSerGlyCysLeuGly                              100105110                                                                     SerThrLeuAlaValMetLysLysSerSerLysPheLeuProValIle                              115120125                                                                     GlyTrpSerMetTrpPheAlaGluTyrLeuPheLeuGluArgSerTrp                              130135140                                                                     AlaLysAspGluLysThrLeuLysTrpGlyLeuGlnArgLeuLysAsp                              145150155160                                                                  PheProArgProPheTrpLeuAlaLeuPheValGluGlyThrArgPhe                              165170175                                                                     ThrProAlaLysLeuLeuAlaAlaGlnGluTyrAlaAlaSerGlnGly                              180185190                                                                     LeuProAlaProArgAsnValLeuIleProArgThrLysGlyPheVal                              195200205                                                                     SerAlaValSerIleMetArgAspPheValProAlaIleTyrAspThr                              210215220                                                                     ThrValIleValProLysAspSerProGlnProThrMetLeuArgIle                              225230235240                                                                  LeuLysGlyGlnSerSerValIleHisValArgMetLysArgHisAla                              245250255                                                                     MetSerGluMetProLysSerAspGluAspValSerLysTrpCysLys                              260265270                                                                     AspIlePheValAlaLysAspAlaLeuLeuAspLysHisLeuAlaThr                              275280285                                                                     GlyThrPheAspGluGluIleArgProIleGlyArgProValLysSer                              290295300                                                                     LeuLeuValThrLeuPheTrpSerCysLeuLeuLeuPheGlyAlaIle                              305310315320                                                                  GluPhePheLysTrpThrGlnLeuLeuSerThrTrpArgGlyValAla                              325330335                                                                     PheThrAlaAlaGlyMetAlaLeuValThrGlyValMetHisValPhe                              340345350                                                                     IleMetPheSerGlnAlaGluArgSerSerSerAlaArgAlaAlaArg                              355360365                                                                     AsnArgValLysLysGlu                                                            370                                                                           (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 295 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      MetAlaMetAlaAlaAlaValIleValProLeuGlyIleLeuPhePhe                              151015                                                                        IleSerGlyLeuValValAsnLeuLeuGlnArgSerGlyCysLeuGly                              202530                                                                        SerAlaLeuAlaValMetLysLysSerSerLysPheLeuProValIle                              354045                                                                        GlyTrpSerMetTrpPheSerGluTyrLeuPheLeuGluArgAsnTrp                              505560                                                                        AlaLysAspGluSerThrLeuLysSerGlyLeuGlnArgLeuAsnAsp                              65707580                                                                      PheProArgProPheTrpLeuAlaLeuPheValGluGlyThrArgPhe                              859095                                                                        ThrGluAlaLysLeuLysAlaAlaGlnGluTyrAlaAlaSerSerGlu                              100105110                                                                     LeuProValProArgAsnValLeuIleProArgThrLysGlyPheVal                              115120125                                                                     SerAlaValSerAsnMetArgSerPheValProAlaIleTyrAspMet                              130135140                                                                     ThrValAlaIleProLysThrSerProProProThrMetLeuArgLeu                              145150155160                                                                  PheLysGlyGlnProSerValValHisValHisIleLysCysHisSer                              165170175                                                                     MetLysAspLeuProGluSerGluAspGluIleAlaGlnTrpCysArg                              180185190                                                                     AspGlnPheValThrLysAspAlaLeuLeuAspLysHisIleAlaAla                              195200205                                                                     AspThrPheAlaGlyGlnLysGluGlnAsnIleGlyArgProIleLys                              210215220                                                                     SerLeuAlaValValLeuSerTrpAlaCysLeuLeuThrLeuGlyAla                              225230235240                                                                  MetLysPheLeuHisTrpSerAsnLeuPheSerSerTrpLysGlyIle                              245250255                                                                     AlaLeuSerAlaLeuGlyLeuGlyIleIleThrLeuCysMetGlnIle                              260265270                                                                     LeuIleArgSerSerGlnSerGluArgSerThrProAlaLysValAla                              275280285                                                                     ProAlaLysProLysAspAsn                                                         290295                                                                        __________________________________________________________________________

We claim:
 1. A recombinant or isolated nucleic acid molecule comprisinga polynucleotide sequence selected from the group consisting of(a) anucleotide sequence encoding the polypeptide having the amino acidsequence in FIGS. 1A-1C (SEQ ID NO: 2) from about amino acid 1 to aboutamino acid 374 in SEQ ID NO: 2 (b) a nucleotide sequence complementaryto a nucleotide sequence encoding the polypeptide having the amino acidsequence in FIGS. 1A-1C (SEQ ID NO: 2) from about amino acid 1 to aboutamino acid 374 in SEQ ID NO: 2; (c) a nucleotide sequence whichhybridizes to the nucleotide sequence in FIGS. 1A-1C (SEQ ID NO: 1)under stringent conditions; and (d) a nucleotide sequence whichhybridizes to the nucleotide sequence that is complementary to thenucleotide sequence in FIGS. 1A-1C (SEQ ID NO: 1) under stringentconditions.
 2. The nucleic acid molecule according to claim 1, whereinsaid polynucleotide sequence encodes an enzyme having membrane-boundacyltransferase activity.
 3. The nucleic acid molecule according toclaim 2, wherein said acyltransferase activity is 2-acyltransferaseactivity.
 4. A microbial host capable of expressing a polynucleotideaccording to claim
 1. 5. A fragment of said polynucleotide according toclaim 1 said fragment comprising at least 15 nucleotides.
 6. Anucleotide sequence encoding an RNA, said RNA being in antisenseorientation to the RNA encoded by the polynucleotide according toclaim
 1. 7. A method for making a recombinant vector comprisinginserting an isolated nucleic acid molecule according to claim 1 into avector.
 8. The nucleic acid molecule according to claim 1, wherein saidpolynucleotide has the complete nucleotide sequence in FIGS. 1A-1C (SEQID NO: 1).
 9. The nucleic acid molecule according to claim 1, whereinsaid polynucleotide has the nucleotide sequence in FIGS. 1A-1C (SEQ IDNO: 1) encoding the polypeptide having the amino acid sequence in FIGS.1A-1C (SEQ ID NO: 2) from about amino acid 1 to about amino acid 375.10. A recombinant vector produced by the method according to claim 7.11. The recombinant vector according to claim 10, wherein said vector isan expression vector containing a promoter which drives the expressionof said nucleic acid molecule.
 12. A method of making a host cellcontaining a recombinant vector comprising introducing the recombinantvector according to claim 10 or 11 into a host cell.
 13. A host cellproduced by the method according to claim 12.